The objective of this study was to determine the survival of Paramecium caudatum, a protozoan ciliate, with different pH under normoxic and hypoxic conditions. P. caudatum was exposed to 18 treatments (in triplicate) of varying pH (4, 4.7, 5.7, 6.7, 7.7, 8.7, 9.7, 10.7 and 11.7) with two different conditions of normoxic and hypoxic. Survival was examined every second day for three weeks. P. caudatum mortality was 100% immediately when exposed to pH 4, 10.7 and 11.7 either in normoxic or hypoxic conditions, whereas at other pH values tested this ciliate could survive mostly under hypoxic conditions. The results suggest that under hypoxic condition, 4.7-6.7 is the best pH range for survival of this species.

One of the most influential factors in determining composition
of freshwater invertebrates` communities including ciliates is pH. An
increase in the rate of photosynthesis by the phytoplankton and other
aquatic plants or the presence of high amount of lime may lead to pH over
9.0 (Wood, 2001). Conversely, pH below 6.0 is due to the high production
of carbon dioxide by aquatic organisms or from the sulfate in the soil,
which yields sulfuric acid (Boyd, 1998).

Freshwater protozoans are dominated at low pH (Packroff,
2000). The richness and abundance of ciliates in natural habitats is also
greater in low pH (4.5) (Mieczan, 2007), even though there is a species-specific
pH tolerance for planktonic freshwater ciliate species (Weisse and Stadler,
2006). Present knowledge of the survival effects of pH on P. caudatum
is generally limited. Most studies that have been reported on Paramecium
and pH largely concern the effect of pH on the forward swimming speed
of the organism (Otter et al., 2005). While the toxicity effect
of extreme values of pH on some species of Paramecium including
species caudatum has been reported (e.g., Rao et al., 2006),
few studies under laboratory conditions performed to simulate those hydrogen
ions that usually occur in the natural habitat. The pH in natural, inland
waters varies from 4.0 to 9.0 (Wetzel, 2001).

When oxygen supply is limited (hypoxia), all organisms
ranging from protozoans to mammals show a protective response known as
an apyrexia (Wood and Malvin, 1991; Malvin et al., 1994; Branco
and Gargaglioni, 2006). In fact, hypoxia results in a decrease in body
temperature and an apyrexia is a behavioural adaptation to hypoxia. An
apyrexia facilitates survival because it lowers metabolic rate of the
organism and increases binding affinity between O2 and its
carrier (Branco and Malvin, 1996). Hypoxia-induced an apyrexia has also
been observed in P. caudatum which is mediated by inhibition of
oxidative phosphorylation (Malvin et al., 1994). Hypoxia can occur
diurnally or seasonally in natural habitats where Paramecium lives
(e.g., pond).

This study was therefore undertaken to examine the laboratory
survival effect of pH on P. caudatum and tolerance of this ciliate
under normoxic and hypoxic conditions. In this study normoxic condition
was defined as air-aerated and a hypoxic condition as a low air-aerated
for the media.

Paramecium is a small microscopic unicellular
organism that is plentiful in freshwater habitats. Many physiological
mechanisms hard to understand in higher vertebrates can be inferred using
Paramecium. The cell surface of Paramecium has been used
as GABA receptors in modulating inhibitory synaptic transmission in neurons
(Ramoino et al., 2006). The thermoregulatory system of the unicellular
Paramecium is much simpler than that of vertebrates. This is largely
due to the extreme complexity of vertebrate thermoregulatory systems.
For example, in Paramecium caudatum, hypoxia appears to exert its
thermoregulatory effects by inhibiting oxidative phosphorylation and decreases
in intracellular (ATP) and pH may be important intermediate signals (Malvin,
1998).

MATERIALS AND METHODS

The experiment was carried in May 2007 at the Biology
department, University of Shahrekord, Shahrekord, Iran. Stock culture
of P. caudatum was grown in a wheat medium bacterised with Klebsiella
pneumoniae under a photoperiod of 12:12 h light:dark (12L:12D) at
room temperature (22-24°C).pH of the stock solution was set at 7.7.
The ciliates were kept in exponential growth. The experimental tests to
ascertain the effect of pH on Paramecium were performed in the
following. P. caudatum was subjected to various pH values. Therefore,
nine treatments of different pH solutions i.e., 4, 4.7, 5.7, 6.7, 7.7,
8.7, 9.7, 10.7 and 11.7 were made by adding acetic acid or sodium carbonate
to the stock solution. Six 50 mL test tubes were selected for each treatment
and filled (15 mL) from the stock culture. The caps of the three test
tubes of any treatment were left open (normoxic or aerated) and the others
were covered by means of parafilm to induce a hypoxia condition. Following
Pedersen and Hansen (2003) to avoid shock effect of pH, the pH was raised
or lowered stepwise (0.5-1 unit at 12 h intervals) until the desired level
was reached. pH was measured using a pH meter, sensitivity 0.01 and a
2 points calibration. Due to shift in pH with time, the pH of each treatment
was adjusted by addition of small amounts of 0.1 mol-1 acetic
acid or sodium bicarbonate. The experiment lasted for three weeks and
sampling was made every 24 h following Salvado and Gracia (1993). Thus
sub-samples (20 μL each) of each tube were taken with a micropipette
and the numbers of live P. caudatum were counted under an optical
microscope with a magnification of 1000x. A highly viscous medium containing
0.5% methylcellulose (Iwadate et al., 1997) was used to slow P.
caudatum swimming and therefore facilitate their counting. A paired
t-test was used to compare the survival effect of varying pH under normoxic
and hypoxic conditions. Significant differences were accepted at p<0.05.

RESULTS

P. caudatum can resist and survive under various acidic and alkaline
situations even though its survival appears to be at more acidic and hypoxic
environment. At pH values of less than 4.7 or greater than 9.7, whether
in normoxic or hypoxic conditions, P. caudatum immediately died,
whereas at pH 4.7-9.7 were able to survive. No significant differences
were found in the cells at pH values of 4.7, while there were significant
differences at pH values of 5.7-9.7 between normoxic and hypoxic conditions
(p<0.05,Table 1,Appendix). During the

Fig. 1:

No. of live Paramecium caudatum
at different pH under normoxic ()
and hypoxic () conditions.
Symbols represent means±SE

Appendix:

Paired samples test

Table 1:

A paired t-test for comparing the survival effect
of varying pH under normoxic (n) and hypoxic (h) conditions

first week of the experiment, the growth rates were not
significantly influenced under normoxic and/or hypoxic condition. The
ciliates survived properly. However, the live number of cells mL-1
declined with time so that there was a significant reduction in the number
of live cells at the end of the second week. The rate of this phenomenon
(a rapid reduction in number of live cells or extinction) was promoted
under normoxic rather than hypoxic condition in particular at pH more
than 7.7, so that at the end of the third week nearly all the cells became
extinct under normoxic condition.

The cell reduction at pH value of 4.7 did not significantly differ throughout
the experiment under either normoxic and/or hypoxic condition so that
a parallel reduction in the number of Paramecium caudatum was found.
The process of divergence in the cell number reduction between normoxic
and hypoxic condition appeared to have taken placed from pH 5.7 onward.
This was evident in particular at pH 7.7 (on day 9 onward) until the end
of the experiment. Except for some days (e.g., day 7 at pH 5.7 or day
8 at pH 8.7) the number of ciliates under normoxic condition almost always
was less than those under hypoxic condition. In effect, P. caudatum
died more quickly under normoxic than hypoxic condition particularly at
higher pH values. Indeed, the higher the pH value, the shorter the life
expectancy. For example at pH 9.7, the ciliates died at a higher rate
than at pH 6.7 (Fig. 1).

DISCUSSION

This study is the first to report some data on the effect
of two environmental factors i.e., pH and normoxic/hypoxic conditions
on the survival of P. caudatum, showing that while this ciliate
protozoan has a wide pH range of tolerance from 4.7 to 9.7, it prefers
more acidic pH (4.7-6.7) and its survival is higher in hypoxic than normoxic
condition. Freshwater protozoans are dominated at low pH (Packroff, 2000)
and the better survival of P. caudatum under acidic condition of
this study may be parallel with an intracellular pH of 6.80 in this species
(Umbach, 1982).

One of the major environmental factors of freshwater
ecosystem is pH. It is impacted by biological processes such as photosynthesis
or respiration (Weisse and Stadler, 2006). The rapid death of P. caudatum
at pH 4, 10.7 and/or 11.7 of this study may be consistent with Doughty
(1986) study in which the P. caudatum were almost immediately immobilized
at pH values of <4.0 or >10.0. In addition, as with the natural,
inland waters of ciliate habitats in which the pH is rarely outside the
range of 4.0-9.0 (Reid, 1961; Nyberg, 1974; Wetzel, 2001), this study
also shows that P. caudatum have maximum (9.7) and minimum tolerances
(4.7) and optimal ranges (4.7-6.7) of pH for survival. Nevertheless, the
wide pH range of tolerance for P. caudatum which is promoted under
hypoxia has ecological consequences. As P. caudatum is amongst
the dominant species of protozoan in natural habitats of ponds (Tharavathi
and Hosetti, 2003), with diurnal or seasonal hypoxia, their ability to
survive in different ranges of pH under hypoxia condition could be an
adaptation to the extreme environment where these species are found. Indeed,
the relative survival abilities of protozoa will be determined by the
rate and timing of declines in their population numbers and by the periodicity
of resource fluctuation that can be tolerated (Jackson and Berger, 1984).

It has been shown that survival in ciliates is related
to ability to form food vacuoles (FVs), but usually in mixed cultures
and due to the competition. For example, survival or extinction of Paramecium
multimicronucleatum cultured with Paramecium tetraurelia is
associated with its ability or inability to form food vacuoles (Maruyama
and Takagi, 1997). This could be ruled out in the current study as just
one type of the Paramecium was originally used. In addition, the
better survival of Paramecium under low pH and/or hypoxic condition
may be related to the secretion of substances which promoted survival
because it appears that ciliates including Paramecium are able
to secrete growth-factor-like substances into the medium (Tanabe et
al., 1990; Christensen and Rasmussen, 1992; Takagi et al.,
1993; Christensen et al., 1995; Vallesi et al., 1995; Tokusumi
et al., 1996) and they function as survival factors (Christensen
and Rasmussen, 1992; Wheatley et al., 1993; Christensen et al.,
1995).

Regardless of normoxic/hypoxic condition, a reduction
in number of the cells took place within the second week when P. caudatum
was maintained at high pH value particularly at 9.7. This finding is in
agreement with Pedersen and Hansen (2003) study who found that exposure
to high pH (>9 or higher) for several days to weeks will result in
significance decrease in number of protozoan. That maintaining P. caudatum
only at pH 4.7 rather the other pH values showed no significance difference
(p = 0.423) suggests that at acidic extremes of pH, the effect of pH is
much far than normoxic/hyopix effect. From pH 4.7 to 9.7 a significance
difference was found under normoxic/hypoxic condition (p<0.05). This
shows that at pH values higher pH than 4.7, condition of maintenance the
cells regarding normoxic/hypoxic situations became important in survival
of this ciliate.

The survival of living systems depends on their ability
to respond appropriately to new situations (Gutierrez et al., 2001).
P. caudatum of this study was able to survive at pH 4.7-9.7 while
it was promoted under hypoxic condition. This could be a compensatory
response in P. caudatum in the current study like an apyrexia which
is protective response and facilitates survival (Malvin et al.,
1994; Branco and Malvin, 1996). As an apyrexia has already been reported
in P. caudatum (Malvin et al., 1994), therefore the observed
hypoxia-induced an apyrexia in P. caudatum may have been led to
survival of this species. As it appears that P. caudatum rarely
encyst under deteriorating conditions including pH change, their ability
to survive for long periods of time under hypoxic condition suggests that
these protozoa utilize the other survival strategies (Jackson and Berger,
1984). In fact, the improved survival of P. caudatum under hypoxic
condition may be simply due to a decrease in respiratory and growth rates.

This study tested the effect of constant external pH
on survival of P. caudatum. Due to shift in pH with time, particularly
for the hypoxic test tubes which were driven upward, I had to add small
amounts of acetic acid or sodium bicarbonate into the solution in order
to maintain a desired pH. pH variation in medium affects not only the
rate of uptake of CO2 but also membrane transport processes
and metabolic functions involved in the biochemical composition, the rate
of metabolic processes and the extracellular production (Raven, 1980;
Nimer et al., 1994; Taraldsvik and Myklestad, 2000). It is obvious
that the enzymes responsible for metabolic pathways in the cells have
different pH optima, implying that a shift in internal pH may affect the
rate metabolic processes (Ouellet and Benson, 1951). Since these effects
are equilibrium-controlled ion fluctuations (Pedersen and Hansen, 2003)
this could explain the significance difference between survival of the
cells particularly at high pH.

In fishes, tolerance to low pH is related to their ability
in maintaining ion balance (Gonzalez, 1998) while in aquatic protist tolerance
to pH has been suggested to heat-shock proteins (Gerloff-Elias et al.,
2005). Therefore, further direction of this research would be to determine
the function and nature of these proteins. In addition, in this study
the desired pH values were reached and maintained by means of chemicals.
However, pH and oxygen condition of natural freshwater habitats rarely
remain constant and may fluctuate diurnally. Thus some other studies need
to be conducted in situ to elucidate the various effects of these
environmental factors on Paramecium survival.

ACKNOWLEDGMENT

The author wishes to express his sincere gratitude to
Mr. Seydaee who helped me in making and maintaining stock solution.

REFERENCES

Boyd, C.E., 1998. Water quality for pond aquaculture. Research and Development (International Center and Aquaculture and Aquatic Environments, Auburn University, Alabama), pp: 1-37.